Conditional (heat-sensitive) mutations remain the most valuable genetic tool available for the in vivo investigation of essential gene requirements, and C. elegans is unique as an animal model in which one can feasibly isolate large numbers of rare conditional mutations. This proposal seeks to expand our identification of conditional mutations in essential C. elegans genes by (i) exploring previously ignored mutant phenotypes, and (ii) developing next generation DNA sequencing-based approaches to greatly reduce the time and labor required to map and positionally clone mutant C. elegans loci isolated after chemical mutagenesis screens. Specific Aim 1 focuses on the identification of 2000 new temperature-sensitive, embryonic-lethal C. elegans mutants; the systematic classification of all mutants into several phenotypic categories; the distribution of multiple mutant classes to collaborators; and the isolation of a large collection of mutants with previously unexplored gonad morphogenesis-defective phenotypes. Specific Aim 2 focuses on developing an Illumina DNA sequencing-based genome-wide approach to Single Nucleotide Polymorphism (SNP) mapping called Restriction-site Associated DNA polymorphism (RAD) mapping. To our knowledge, we are the first laboratory to explore applying this recently developed technology to the mapping of mutant loci in C. elegans. It promises to provide a high throughput and cost-effective approach to the mapping and positional cloning of large numbers of mutant loci. Specific Aim 3 explores Illumina DNA sequencing-based whole genome sequencing to identify the mutational lesions responsible for conditional embryonic-lethality in the mutants isolated in Aim 1 and mapped to small intervals in Aim 2. Our ultimate goal is to greatly expand the use of chemical mutagenesis screens to identify conditional mutations in essential C. elegans genes. By developing high throughput approaches to the positional cloning of conditionally mutant loci, our proposed exploratory research will substantially impact research by laboratories throughout the world that use C. elegans as an animal model for investigating many different and fundamentally important biological processes. PUBLIC HEALTH RELEVANCE: The nematode Caenorhabditis elegans is unique as an animal model in which one can feasibly isolate large numbers of rare conditional mutations in essential genes. Conditional (heat-sensitive) mutations remain the most valuable genetic tool available for the investigation of essential gene requirements, which often have multiple essential requirements, in many cases even during a single cell division cycle. Moreover, essential C. elegans genes are with few exceptions widely conserved in other organisms including humans, and are in many cases of direct relevance to our understanding of and ability to detect and treat cancers and other important human diseases. By promoting the isolation of large collections of these valuable genetic tools for the study of conserved and essential genes, we will contribute substantially to our basic understanding of numerous fundamental biological processes under investigation in laboratories throughout the world. The investigation of biological processes in model organisms like C. elegans is of fundamental importance for understanding human disease mechanisms, and for identifying possible therapeutic targets and opportunities.